Received: 2 May 2020 | Revised: 31 October 2020 | Accepted: 17 November 2020 DOI: 10.1002/mgg3.1569 ORIGINAL ARTICLE Early truncation of the N-terminal variable region of EYA4 gene causes dominant hearing loss without cardiac phenotype Yanfang Mi1 | Danhua Liu2,8 | Beiping Zeng3,4 | Yongan Tian3,4 | Hui Zhang5 | Bei Chen1 | Juanli Zhang6 | Hong Xue7 | Wenxue Tang8,2,4 | Yulin Zhao1 | Hongen Xu2 1Department of Otorhinolaryngology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2Precision Medicine Center, Academy of Medical Science, Zhengzhou University, Zhengzhou, China 3BGI College, Zhengzhou University, Zhengzhou, China 4Henan Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China 5Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 6Henan Province Medical Instrument Testing Institute, Zhengzhou, China 7Sanglin Biotechnology Ltd, Zhengzhou, China 8The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, China Correspondence Yulin Zhao, Department of Abstract Otorhinolaryngology, the First Affiliated Background: Autosomal dominant hearing loss (ADHL) accounts for about 20% of Hospital of Zhengzhou University, all hereditary non-syndromic HL. Truncating mutations of the EYA4 gene can cause Jianshedong Road No.1, Zhengzhou 450052, China. either non-syndromic ADHL or syndromic ADHL with cardiac abnormalities. It has Email: [email protected] been proposed that truncations of the C-terminal Eya domain lead to non-syndromic Hongen Xu, Precision Medicine Center, HL, whereas early truncations of the N-terminal variable region cause syndromic HL Academy of Medical Science, Zhengzhou with cardiac phenotype. University, Daxuebei Road No. 40, Zhengzhou 450052, China. Methods: The proband and all the other hearing impaired members of the family un- Email: [email protected] derwent a thorough clinical and audiological evaluation. The cardiac phenotype was examined by ECG and echocardiography. Their DNA was subjected to target exome Present address Yanfang Mi, The Second Affiliated sequencing of 129 known deafness genes. The sequencing data were analyzed and Hospital of Zhengzhou University, the candidate variants were interpreted following the ACMG guidelines for clinical Zhengzhou, China sequence interpretation. The effect of candidate variant on EYA4 gene expression was Funding information assessed by quantitative PCR and western blot of gene production in blood. Collaborative Innovation Project of Results: We report a Chinese family cosegregating post-lingual onset, progressive Zhengzhou (Zhengzhou University), ADHL with a novel nonsense mutation NM_004100.4:c.543C>G (p.Tyr181Ter) of Grant/Award Number: 18XTZX12004; Medical Science and Technology EYA4. Two affected members show no cardiac abnormalities at least until now re- Projects in Henan, Grant/Award Number: vealed by electrocardiography and echocardiography. The overall expression level of SBGJ2018043; Medical Science and EYA4 Technology Research Project Joint the gene in the proband was lower than that in his unaffected relative. Construction Project in Henan, Grant/ Conclusion: This report expands the mutational spectrum of the EYA4 gene and high- Award Number: SBGJ2018020152 lights the fact that more data are needed to elucidate the complex genotype–pheno- type correlation of EYA4 mutations. Yanfang Mi and Danhua Liu are Joint first authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC. Mol Genet Genomic Med. 2021;9:e1569. wileyonlinelibrary.com/journal/mgg3 | 1 of 9 https://doi.org/10.1002/mgg3.1569 2 of 9 | MI ET AL. KEYWORDS autosomal dominant hearing loss, EYA4, next-generation sequencing 1 | INTRODUCTION genetic cause was investigated using targeted sequencing of 129 known deafness genes. A novel nonsense variant, Hearing loss is the most common sensory defect in humans. NM_004100.4:c.543C>G (p.Tyr181Ter) in exon 8 of the EYA4 The prevalence of permanent HL at birth is 1.07/1000 (Morton gene, was identified in the family. The onset age and audio- & Nance, 2006). HL is a heterogeneous disorder caused by ge- grams were compared with other studies, and the impact of netic and environmental factors, yet half of the cases tend to be reported mutations on cardiac phenotype has been discussed. genetic (Nance, 2003). If the pathology is genetically related, This study expands the mutational spectrum of the EYA4 gene it can be classified according to the pattern of inheritance (au- and suggests that next-generation sequencing is effective in elu- tosomal dominant, autosomal recessive, and X-linked), and cidating the etiology of familiar progressive HL. the presence (syndromic) or absence (non-syndromic) of ab- normalities in other parts of the body. It has been estimated that approximately 80% of genetic deafness is non-syndromic, 2 | MATERIALS AND METHODS where 80% of non-syndromic HL have been inherited in an au- tosomal recessive manner (Morton & Nance, 2006). In contrast 2.1 | Ethical compliance to the autosomal recessive HL, which is congenital and pre- lingual, patients with autosomal dominant non-syndromic HL This study was conducted according to the Helsinki (ADNSHL) often have a family history of post-lingual HL. Declaration and approved by the local institutional ethics So far, a total of 47 genes have been identified as causal committee in human research of Zhengzhou University (ref- genes for ADNSHL (http://hered itary heari ngloss.org), including erence no. 2018008). Written informed consent for genetic DIAPH1 (Lynch et al., 1997), KCNQ4 (Kubisch et al., 1999), testing and the submission of the study was obtained from all and GJB3 (Xia et al., 1998). Autosomal dominant deafness the studied individuals. 10 (DFNA10, OMIM #601316) locus was first identified and mapped on chromosome 10 in 1996 in a large American family as a causal gene for ADNSHL (O'Neill et al., 1996). In 2001, Wayne 2.2 | Subjects and clinical evaluation et al. identified nonsense mutations in the EYA4 gene (OMIM: 603550) in two unrelated families segregating at DFNA10, and The proband was a 28-year-old male Chinese, who was re- defined EYA4 (Eyes Absent Homolog 4) as the causative gene for ferred to the Department of Otolaryngology at the Second DFNA10 (Wayne et al., 2001). The EYA4 gene is composed of 21 Affiliated Hospital of Zhengzhou University in Zhengzhou. exons encoding a 640 amino acid protein, which contains a highly Detailed family history questioning revealed the other four conserved region (eya homologous region, eyaHR) and a more affected members across three generations with AD inherit- divergent proline-serine-threonine (PST)-rich transactivation do- ance patterns (Figure 1). All affected family members under- main (eya variable region, eyaVR) (Borsani et al., 1999). EYA4 went a medical history interview that included questions on is a member of the vertebrate eya gene family, a group of tran- onset age and degree of HL, other clinical manifestations, scriptional activators that interact with other proteins to ensure and environmental factors (viral infection, noise, and oto- the healthy development of multiple organs, including the eye, toxic drugs). A complete clinical evaluation, audiological muscle, kidney, inner ear, and heart (Wang et al., 2008; Wayne tests, and cardiac examination were performed in the affected et al., 2001). Although some mutations in EYA4 cause syndromic family members who agreed to participate in this study. The HL with dilated cardiomyopathy (DCM) as described in a fam- level of HL was classified into four tiers in terms of pure ily (Schonberger et al., 2000, 2005) and a Japanese patient (Abe tone average (averaged over 0.5, 1.0, 2.0, and 4.0 kHz): mild et al., 2018), the majority of mutations in this gene are associated (21–40 dB), moderate (41–70 dB), severe (71–90 dB), and with ADNSHL. Hearing impairment for EYA4 mutations is char- profound (>91 dB). acterized by post-lingual and progressive sensorineural HL. To date, 34 diseases causing EYA4 mutations have been described, including four nonsense mutations, 12 missense mutations, nine 2.3 | Target exome sequencing of known small indels, six splicing mutations, and three gross deletions deafness genes (Stenson et al., 2012). In this study, we report on a case of a three-generation Genomic DNA was isolated from peripheral blood leuko- family with late-onset progressive sensorineural HL. The cytes using the GenMagBio Genomic DNA Purification kit MI ET AL. | 3 of 9 FIGURE 1 Pedigree of the family with non-syndromic autosomal dominant sensorineural hearing loss segregating EYA4 c.543C>G (canonical transcript NM_172105.3.). (a) Family pedigree. Open symbols unaffected; filled black symbols affected; diagonal line deceased. (b) Sanger sequencing. The red arrow indicates the EYA4 c.543C>G mutation (GenMagBio, Changzhou, China) following the manufac- steps described above were performed in the framework of turer's standard procedures. Genomic DNA from affected bcbio-nextgen (https://github.com/bcbio/ bcbio -nextgen), and unaffected members was fragmented to an average size which provides best practice pipelines for variant calling, an- of 250 bp, and end repair, adapter ligation as well as